Project Summary Embryonic definitive hematopoiesis is initiated by endothelial-hematopoietic transition (EHT) in which endothelial cells alter fate to become hematopoietic progenitors and stem cells. In mammals there are two major sites of EHT, the yolk sac vasculature and the major arteries of the embryo (dorsal aorta, vitelline, umbilical). Although the process of EHT is highly conserved and required for definitive hematopoiesis in all vertebrates, the signals that control the conversion of vascular endothelial cells to hemogenic endothelial cells are poorly understood. Recent studies have implicated hemodynamic shear forces and inflammatory cytokines in promoting EHT, but how these diverse signals stimulate the formation of hemogenic endothelium remains unknown. We have previously demonstrated that the MEKK3 MAPK pathway mediates endothelial cell responses to both inflammatory cytokines and hemodynamic shear forces, in large part by increasing expression of the KLF2 and KLF4 transcription factors. Our preliminary studies demonstrate that endothelial loss of MEKK3 or KLF2+KLF4 results in lethal fetal anemia and failure of EHT in both the yolk sac and embryo. This proposal will test the hypothesis that MEKK3-KLF2/4 signaling integrates endothelial cell stimulation by cytokines and fluid forces to trigger EHT and the onset of definitive hematopoiesis. These studies are expected to yield new insight into the signals that initiate EHT, findings that may be used to generate new hematopoietic stem and progenitor cells for therapeutic purposes later in life.